Antagonist peptides to VEGF receptor Flt-1

ABSTRACT

The present invention provides a polypeptide or a pharmacologically acceptable salt thereof comprising one or more, identical or different, amino acid sequences of A-W—H—X1-D-X2-X3-X4-W—W—X5-X6-X7-X8-B (the meaning of each symbol in the formula is defined in the description) and a pharmaceutical composition for treating an angiogenic disease such as a solid tumor comprising said polypeptide. The present invention also relates to a polynucleotide encoding the above-mentioned polypeptide and use of the polynucleotide in gene therapy.

FIELD OF THE INVENTION

The present invention relates to peptides acting as antagonists to VEGFreceptor Flt-1 and useful in the treatment of angiogenic diseases. Thepresent invention also relates polynucleotides encoding the peptides,vectors and cells containing the polynucleotides, methods andpharmaceutical compositions for treating angiogenic diseases by usingthe peptides, polynucleotides, vectors or cells.

BACKGROUND OF THE INVENTION

Angiogenesis is essential for normal biological processes includingreproduction, development and wound healing. Although angiogenesis is ahighly regulated process in normal conditions, many diseases are causedby persistent disorders of angiogenesis. Theses diseases are calledangiogenic diseases. That is, the disorders of angiogenesis can causeparticular diseases directly, or exacerbate the existing diseases. Forinstance, the neovascularization in eye is the most common cause ofblindness. In some existing diseases such as arthritis, new-developedcapillaries invade the joint and destroy cartilage. In diabetes,new-developed capillaries invade vitreous, resulting in bleeding andblindness. Growth and metastasis of solid tumor depend onneovascularization which involves the degradation of the basementmembrane of the starting blood vessel, the proliferation and migrationof endothelial cells, and the development of new vessels (Folkman J.Angiogenesis in cancer, rheumatoid and other disease. Nature Med.,1995a, 1, 27-30) etc.

As a member of the family of platelet-derived growth factors, vascularendothelial growth factor (VEGF) is the most direct nitogen of vascularendothelial cells (Ferrara N., Molecular and biological properties ofvascular endothelial growth factor. J. Mol. Med., 1999, 77:527-543). Theactivities of VEGF are mediated by binding to its receptors (VEGFR), andits biological effects are exhibited through endocellular signaltransduction. The binding of VEGF to its receptor Flt-1 (fms-liketyrosine domain containing receptor) can result in endothelial cellmigration, differentiation, and ultimately forming tubular structure(blood vessel rudiment). Flt-1 is an important target for inhibitingangiogenesis (Hanahan H., Signaling vascular morphogenesis andmaintenance. Science, 1997, 277:48-50; Shinichi K. et al., Roles of twoVEGF receptors, Flt-1 and KDR, in the VEGF effects in human vascularendothelial cells. Oncogene, 2000, 19: 2138-2146).

Some peptides acting as antagonists against VEGF binding to its receptorKDR are reported by Binetruy-Tournaire R. et al. in The EMBO Journal.2000, 19(7): 1525-1533. It is said that they can inhibit theneovascularization induced by VEGF.

Due to the fact that the angiogenesis inhibitors clinically availableare not satisfactory in terms of, among others, their severe systemtoxicity (Suramin) or weak activity of anti-angiogenesis (interferon,antiestrogen), therefore new angiogenesis inhibitors are needed.

SUMMARY OF THE INVENTION

The present invention provides a polypeptide having one or more aminoacid sequences of the following formula (I), which may be identical ordifferent, or a pharmaceutically acceptable salt thereof:A-W—H—X1-D-X2-X3-X4-W—W—X5-X6-X7-X8-B  (I)wherein:

A is 0 to 3 optionally protected naturally occurring amino acidresidues;

X1-X5 independently represents a sectuence gap or an optionallyprotected naturally occurring amino acid residues;

X6 represents a sequence gap or an optionally protected naturallyoccurring hydrophobic amino acid residue;

X7 represents a sequence gap or an optionally protected naturallyoccurring amino acid residue;

X8 represents a sequence gap or an optionally protected naturallyoccurring hydrophobic amino acid residue;

B is 0 to 3 optionally protected naturally occurring amino acidresidues;

W represents optionally protected tryptophan;

H represents optionally protected histidine;

D represents optionally protected aspartate.

The present invention also provides a pharmaceutical compositioncomprising one or more peptides of the present invention as describedabove and optionally a pharmaceutically acceptable carrier or excipient.

The present invention relates to a peptide of the present invention as amedicament, especially as a medicament for treating angiogenic diseases.

The present invention also relates to use of one or more peptides of thepresent invention as described above for the preparation of a medicamentfor treating angiogenic diseases.

The present invention also provides a method for treating a subject inneed of anti-angiogenesis treatment, comprising administering to thesubject a therapeutically effective amount of one or more peptides ofthe present invention as described above.

The present invention further relates to a nucleotide sequence encodinga peptide of the present invention as described above.

The present invention also relates to a plasmid or viral vectorcontaining a nucleotide sequence of the present invention as describedabove.

The present invention also relates to a cell containing a nucleotidesequence or a vector of the invention as described above.

The present invention also provides a nucleotide sequence, vector orcell line of the present invention as a medicament.

The present invention also relates to a pharmaceutical composition fortreating angiogenic diseases containing a nucleotide sequence, vector orcell line of the invention as described above.

The present invention further relates to use of a nucleotide sequence orvector of the present invention in preparing, from cells obtained from asubject, cells to be redelivered to said subject for treating angiogenicdiseases.

The present invention relates to a method for treating angiogenicdiseases, which comprises administering to a subject in need of suchtreatment a therapeutically effective amount of a nucleotide sequence,vector or cell of the present invention as described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Results of a radio-immunological competitive inhibition test fordetermining the inhibition of the binding of VEGF to the solublereceptor, sFlt-1, by fusion proteins containing different peptides ofthe present invention.

FIG. 2. Results of an experiment for determining the inhibition of theVEGF-stimulated proliferation of human umbilical vein-derivedendothelial cells by different peptides of the present invention.

FIG. 3. Results of an experiment for determining the inhibition of theproliferation of blood vessels on chick chorioallantoic membrane by apeptide of the present invention: (A) VEGF stimulated the blood vesselsto proliferate significantly; (B) DHFR had no notable effect on theblood vessels; (C) the blood vessels of chick embryo treated by F56 andF90 were derangeable in distribution and atrophic.

FIG. 4. Inhibition of the growth of tumor in nude mice byintraperitoneally injected fusion protein containing of a peptide of thepresent invention. It shows the state of tumor on the 30th day afterwithdrawal.

FIG. 5. Inhibition of the growth of tumor in nude mice byintraperitoneally injected fusion protein containing a peptide of thepresent invention. It shows the growth curve of tumor in nude mice afteradministrating.

DETAILED DESCRIPTION OF THE INVENTION

Based on the strategy of inhibiting angiogenesis by blocking VEGF frombinding to its corresponding receptor Flt-1, the inventor used solubleFtl-1 obtained by cloning and expression as target to screen phagepeptide library. The inventor discovered and proved by in vivo and invitro experiments that the peptides and fusion proteins containing thepeptides as set forth below have anti-angiogenesis activity and caninhibit growth and metastasis of tumor and treat other angiogenicdiseases.A-W—H—X1-D-X2-X3-X4-W—W—X5-X6-X7-X8-B  (I)

wherein:

A is 0 to 3 optionally protected naturally occurring amino acidresidues;

X1-X5 independently represent a sequence gap or optionally protectednaturally occurring amino acid residues;

X6 represents a sequence gap or an optionally protected naturallyoccurring hydrophobic amino acid residue;

X7 represents a sequence gap or an optionally protected naturallyoccurring amino acid residue;

X8 represents a sequence gap or an optionally protected naturallyoccurring hydrophobic amino acid residue;

B is 0 to 3 optionally protected naturally occurring amino acidresidues;

W represents optionally protected tryptophan;

H represents optionally protected histidine;

D represents optionally protected aspartate.

In a preferred embodiment, the peptides of the present invention areunprotected free peptides or pharmaceutically acceptable salts thereof.

In another preferred embodiment, the α amino group on N-terminal of thepeptides of the present invention has an amino protective group, and/orthe α carboxy group on C-terminal has a carboxylic protective group.

Preferably, in the above formula (I), X2 represents a sequence gap orPro.

Preferably, in the above formula (I), X5 is a polar amino acid residue.

Preferably, in the above formula (I), X7 is a hydrophobic amino acidresidue.

In a preferred embodiment, the polypeptide of the present inventioncontains one amino acid sequence of formula (I). In this embodiment,more preferably, the polypeptides of the present invention are:

H₂N-Trp-His-Ser-Asp-Met-Glu-Trp-Trp-Tyr-Leu-Leu-Gly-COOH (F56) SEG IDNO: 1;

H₂N-Trp-His-Val-Asp-Glu-Thr-Trp-Trp-Leu-Leu-Met-Leu-COOH (F87) SEQ IDNO: 2;

H₂N-Trp-His-Asp-Pro-Thr-Pro-Trp-Trp-Ser-Trp-Glu-Ile-COOH (F90). SEQ IDNO: 3.

In another preferred embodiment, the polypeptide of the presentinvention is a fusion protein composed of an-amino acid sequence offormula (I) and another polypeptide. Based on the activities of thepeptides of the present invention, it can be envisaged that peptides ofthe present invention can be fused with other functional peptides orproteins to form multifunctional fusion proteins of different forms. Forexample, it is possible to fuse the peptides of the present inventionwith the following peptides or proteins to form fusion proteins:

-   -   1. Various cytokines, such as interleukin-2, to topically        activate T lymphocytes etc. while blocking the receptors in the        tumor site;    -   2. Soluble receptor KDR or Flt-1, to exhibit double functions of        blocking the receptor and binding VEGF by the soluble receptor,        therefore to inhibit the binding of VEGF to its receptors by        different ways at the same time;    -   3. Specific antibodies or fragments thereof such as tumor        vascular endothelial cell specific antibody or fragment thereof,        to make the in vivo distribution of the peptides of the present        invention specific to a target;

4. Other functional peptides optionally through a linker of certainlength, such as peptides for blocking receptor KDR disclosed in Chinesepatent application 01142203.3 to the present inventor with the samefiling date as the present application, in particular:

H₂N-His-Thr-Met-Tyr-Tyr-His-His-Tyr-Gln-His-His-Leu-COOH (K237) (SEQ IDNO: 4),

H₂N-Met-His-Asn-His-His-Asn-His-Pro-Arg-Pro-Ser-Ser-COOH (K112) (SEQ IDNO: 5), or

H₂N-Cys-Asp-Pro-Leu-Leu-Lys-His-His-Thr-His-Pro-Lys-COOH (K212) (SEQ IDNO: 6).

In this case, one molecule can block two kinds of receptors, KDR andFlt-1, therefore inhibit the binding of VEGF to its receptors moreeffectively;

5. B7 molecule, etc., to activate lymphocytes in the tumor tissuetopically while blocking the receptors;

6. Any other peptides or proteins compatible with and functionallycomplementing or synergizing the peptides of the present invention.

In another preferable embodiment, the polypeptides of the presentinvention contain two or more amino acid sequences of formula (I), beingidentical or different, optionally with a suitable linker between theadjacent amino acid sequences. In other words, the peptides of thepresent invention can be connected with one another optionally throughsuitable linker(s) to form homo-multimers or hetero-multimers.

As used herein, “amino protective group” refers to a chemical groupprotecting an amino group of an amino acid or peptide from a reaction.For amino protective groups, see the description in Greene “ProtectiveGroups In Organic Synthesis” (John Wiley & Sons, New York(1981)). Forexample, the amino protective groups include C₁-C₆-acyl such as acetylgroup, propionyl group, pivaloyl group, tert-butyl acetyl group, etc.;other acyl such as 2-chloro-acetyl group, 2-bromoacetyl group,trifluoro-acetyl group, trichloroacetyl, phthalyl group, benzoyl,4-chloro-benzoyl, 4-bromo-benzoyl, 4-nitro-benzoyl, etc.; sulfonyl suchas benzene-sulfonyl, para-tosyl, etc.; the groups capable of formingcarbamate, such as carbobenzoxy group, para-chloro-carbobenzoxy group,para-methoxy-carbobenzoxy group, para-nitro-carbobenzoxy group,para-bromo-carbobenzoxy group, 3,4-dimethoxy-carbobenzoxy group,3,5-dimethoxy-carbobenzoxy group, 2,4-dimethoxy-carbobenzoxy group,4-methoxy-carbobenzoxy group, 2-nitro-4,5-dimethoxy-carbobenzoxy group,3,4,5-trimethoxy-carbobenzoxy group, 1-(biphenylyl)-1-methylethoxycarbonyl group, α,α-dimethyl 3,5-dimethoxy carbobenzoxy group,diphenylmethoxycarbonyl group, t-butoxycarbonyl, diisopropylmethoxycarbonyl group, isopropoxy-carbonyl group, ethoxycarbonyl group,methoxycarbonyl group, allyloxycarbonyl group,2,2,2-trichloroethoxycarbonyl group, phenoxycarbonyl group,4-nitrophenoxycarbonyl group, flourenyl-9-methoxycarbonyl group,cyclopentyloxy-carbonyl group, adamantyloxy-carbonyl group,cyclohexyloxy-carbonyl group, phenylthiocarbonyl group; aralkyl groupsuch as benzyl, trityl, carbobenzoxy group, 9-flourenyl-methoxycarbonylgroup (Fmoc), etc.; and silyl such as trimethylsilyl, etc. The preferredamino protective groups are formyl group, acetyl group, benzoyl group,pivaloyl group, tert-butyl acetyl group, benzenesulfonyl group, benzyl,tert-butoxycarbonyl group (Boc), carbobenzoxygroup (Cbz). For example,the α amino group of lysine is protected by an acid-labile group (suchas Boc), while the ε amino group is protected by an alkali-labile group(such as Fmoc) so that a selective de-protection is possible insynthesis.

As used herein, a “carboxy protective group” refers to an ester or amidogroup protecting a carboxy group while functional group(s) on othersite(s) is/are under reaction. For carboxy protective groups, see thedescription in Greene “Protective Groups In Organic Synthesis” (JohnWiley & Sons, New York (1981)). For example, the carboxy protectivegroups include C₁-C₈ lower alky such as methyl, ethyl, tert-butyl, etc.;aralkyl groups such as phenethyl, benzyl, or substituted benzyl; arylalkenyl such as phenylvinyl; dialkylaminoalkyl such asdimethylaminoethyl, etc. alkanoyloxyalkyl such as acetoxymethyl,butyroxymethyl, valeryloxymethyl, isobutyroxymethyl,isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl, 1-(pivaloyloxy)-1-ethyl,1-methyl-1-(propionyloxy)-1-ethyl, pivaloyloxymethyl,propionyloxymethyl, etc.; cycloalkanoyloxyalkyl such ascyclopropylcarbonyloxymethyl, cyclobutylcarbonyloxymethyl,cyclopentylcarbonyloxymethyl, cyclohexylcarbonyloxymethyl, etc.;arylcarbonyloxyalkyl such as benzoyloxymethyl, benzoyloxyethyl, etc.;araklycarbonyloxyalkyl such as benzylcarbonyloxymethyl,2-2-benzylcarbonyloxyethyl, etc.; alkyoxycarbonylalkyl orcycloalkyoxycarbonylalkyl such as methoxycarbonylmethyl,cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl, etc.;alkyoxycarbonyloxyalkyl or cycloalkyoxycarbonyloxyalkyl such asmethoxycarbonyloxymethyl, tert-butoxycarbonyloxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl, etc;aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,2-(5-indanyloxycarbonyloxy)ethyl, etc.; alkyoxyalkylcarbonyloxyalkylsuch as 2-(1-methoxy-2-methyl-2-propionyloxy)ethyl, etc.;arylalkyoxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl, etc.;arylalkenoxycarbonyloxyalkyl such as 2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl, etc.; alkyoxycarbonylaminoalkyl such astert-butyoxycarbonylaminomethyl, etc.; alkylaminocarbonylaminoalkyl suchas methylaminocarbonylaminomethyl, etc.; alkanoylaminoalkyl such asacetaminomethyl, etc.; heterocyclylcarbonyloxyalkyl such as4-methylpiperazinylcarbonyloxymethyl, etc.; dialkylaminocarbonylalkylsuch as dimethylaminocarbonylmethyl, diethylaminocarbonylmethyl, etc.The preferred carboxy protective groups are C₁-C₆ lower alkyl, C₃-C₇cycloalkyl or aralkyl ester, such as methyl ester, ethyl ester, propylester, isopropyl ester, butyl ester, sec-butyl ester, isobutyl ester,pentyl ester, isopentyl ester, cyclohexyl ester, phenethyl ester, etc.;or alkanoyloxycarbonyl, cycloalkanoyloxycarbonyl, arylcarbonyloxyalkylor aralkylcarbonyloxyalkyl ester. For example, the α carboxy group ofaspartate is protected by acid-labile group (such as tert-butyl), whilethe β carboxy group is protected by hydrogenation-labile group (such asbenzyl) so that a selective de-protection is possible in the process ofsynthesis.

The naturally occurring amino acids (termed “genetically encoded”) asused herein include polar amino acids: serine (Ser), threonine (Thr),cysteine (Cys), tyrosine (Tyr), aspartate (Asp), asparagine (Asn),glutamate (Glu), glutamine (Gln), lysine (Lys), arginine (Arg) andhistidine (His); hydrophobic amino acids: glycine (Gly), alanine (Ala),valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro),phenylalanine (Phe), tryptophan (Try) and methionine (Met). Unlessotherwise indicated, the stereochemistry of the α carbon in said aminoacids and the amino acid residues of peptides of the present inVentionis the their natuial configuration, i.e., “L” configuration. Of course,this does not apply to a non-chiral amino acid.

Said amino acids of the present invention may have non-natural sidechain residues, as in homo-phenylalanine, phenyiglycine, norvaline,norleucine, ornithine, thiazolyl alanine, etc.

The peptides of the present invention can be synthesized by conventionalmethods of solid phase peptide synthesis known to those skilled in theart. For example, the peptides of the present invention can besynthesized by solid phase chemistry techniques according to theprocedures described by Steward and Young (Steward, J. M. and Young, J.D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Company,Rockford, Ill., (1984)) using an Applied Biosystem synthesizer or aPioneer™ peptide synthesizer. Alternatively, multiple fragments may besynthesized then linked together to form larger fragments. For solidphase peptide synthesis, many related techniques are disclosed inSteward, J. M. and Young, J. D., Solid Phase Peptide Synthesis, W. H.Freeman Co. (San Francisco), 1963; and J. Meienhofer, Hormonal Proteinsand Peptides, vol. 2, p.46, Academic Press (New York), 1973. Generally,these methods include sequential addition of one or more amino acids orsuitably protected amino acids to a growing peptide chain. Normally,either the amino group or the carboxy group of the first amino acid isprotected by a suitable protective group. Then the protected amino acidis attached to an inert support of solid phase, the next amino acid ofthe sequence with a suitably protected complementary amino group orcarboxy group is added under the conditions suitable for forming theamide linkage. Then, the protective group is removed from the newlyadded amino acid residue, and the next amino acid that is suitablyprotected if required is added. The process is repeated until all thedesired amino acids are linked in proper sequence. Any remainingprotective groups and any solid support are removed sequentially orconcurrently to give the final polypeptide. More than one amino acid canbe added to the growing peptide chain at a time by simple modificationto the above general procedure.

A particularly preferred method for preparing the peptides of thepresent invention involves solid phase peptide synthesis wherein theamino acid α amino group is protected by an acid or base sensitiveprotective group. The protective groups should be stable underconditions of forming peptide linkage but easy to remove withoutdestructing the growing peptide chain and racemizing any of the chiralcenter contained therein. Suitable protective groups include 9-flourenylmethoxycarbonyl group (Fmoc), tert-butoxycarbonyl group (Boc),carbobenzoxy group (Cbz), 2-cyano-tert-butoxycarbonyl group, etc.9-flourenyl methoxycarbonyl group (Fmoc) is particularly preferred forsynthesizing the peptides of the present invention.

Other preferred side chain protective groups are2,2,5,7,8-pentamethylchroman-6-sulfonyl (pmc), nitro, p-tosyl,4-methoxybenzene-sulfonyl, Cbz, Boc and adamantyloxycarbonyl for sidechain amino group of lysine and arginine; benzyl, o-bromo-carbobenzoxygroup, 2,6-dichlorobenzyl, isopropyl, tert-butyl, cyclohexyl,cyclopentyl and acetyl for tyrosine; tert-butyl, benzyl andtetrahydropyranyl for serine; trityl, benzyl, Cbz, p-tosyl and2,4-dinitophenyl for histamine; formyl for tryptophan; benzyl andtert-butyl for aspartate and glutamate; trityl for cysteine.

In the method of solid phase peptide synthesis, first of all, theC-terminal α amino acid has to be attached to a suitable solid supportor resin. The solid supports for the above-mentioned method are thosematerials that are inert to the reagents and reaction conditions of thestepwise condensation-deprotection reactions and insoluble in thesolvents used. The preferred solid support for synthesis of α C-terminalcarboxyl peptides is 4-hydroxymethylphenoxy-copoly (styrene-1%divinylbenzene). The preferred support of solid phase for synthesis of αC-terminal amide peptides is4-(2′,4′-dimethoxyphenyl-Fomc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Generally, theC-terminal α amino acid is coupled to the resin according to thefollowing method: the α amino acid is coupled with the resin for about 1to 24 hours at 10 to 50° C. in appropriate solvent such asdichloromethane or DMF by means of N,N′-dicyclohexylcarbodiimide (DCC),N,N′-diisopropylcarbodiimide (DIC) orO-benzotriazole-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazole-1-yloxy-tri(dimethylamino)phosphonium hexafluorophosphate(BOP) or di-(2-oxo-3-oxazolidinyl) phosphine chloride (BOPCI). When thesolid support is 4-(2′,4′-dimethoxyphenyl-Fomc-aminomethyl)phenoxylacetamidoethyl resin, the Fmoc group should be removed by a secondaryamine (preferably piperidine) before coupling the C-terminal α aminoacid according to the above-mentioned procedure. The preferred methodfor coupling with the deprotected4-(2′,4′-dimethoxyphenyl-Fomc-aminomethyl)phenoxy acetamidoethyl resinis to use O-benzotriazole-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole(HOBT, 1 equivalent) in DMF.

The subsequent coupling of amino acids can be performed in an automaticpeptide synthesizer well known in the art. In a preferred embodiment,the α N-terminal amino acids of the growing peptide chain is protectedby Fmoc. Protective group Fmoc can be removed from α N-terminal side ofthe growing peptide chain by a secondary amine (preferably piperidine).Each protected amino acid is added in about 3-fold molar excess. Thecoupling reaction is preferably performed in DMF. Generally the couplingagents are O-benzotriazole-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 1 equivalent) and 1-hydroxybenzotriazole(HOBT, 1 equivalent). At the end of the solid phase synthesis, thepeptide is removed from the resin and deprotected, either successivelyor concurrently. The resin-polypeptide complex can be treated with acleavage reagent containing thianisole, water, ethanedithiol andtrifluoroacetic acid to carry out the removal of polypeptide and thedeprotection simultaneously. The polypeptide may be removed from theresin by aminolysis with an alkylamine, if the α C-terminal of thepolypeptide is an alkylamide. Alternatively, the polypeptide can beremoved by transesterification (e.g., with methanol) followed byaminolysis or by direct transamidation. The protected polypeptide socleaved can be purified or applied in the next step withoutpurification. The protective groups of side chain can be removed bycleavage mixture set forth above. The fully deprotected polypeptide canbe purified by using some or all types of the following chromatographicmethods: ion exchange on a weakly basic resin (for example acetateform); hydrophobic adsorption chromatography on an underivitizedpoly(styrene-divinylbenzene) resin (for example Amberlite XAD); silicagel adsorption chromatography; ion exchange chromatography oncarboxymethylcellulose; partition chromatography, e.g., on SephadexG-25, LH-20, or countercurrent distribution; high performance liquidchromatography (HPLC), especially reverse-phase HPLC on a column ofoctyl- or octadecyl silica. The molecular weight of synthesizedpolypeptides may be determined by fast atom bombardment (FAB) massspectrum. For the specific synthesis of the polypeptides of the presentinvention, see examples 1 to 3.

Long polypeptides and fusion proteins of the present invention arepreferably obtained by expression of a polynucleotide containing thecoding sequence of the polypeptide or fusion protein of the presentinvention in a suitable host and purification of the so-expressedpolypeptide. Both sense and antisense DNA strands of the nucleotidesequences encoding the peptides of the present invention can besynthesized by phosphite triester method well known in the art on a DNAsynthesizer. The oligonucleotides encoding the peptides of the presentinvention may be obtained by annealing the synthesized complementarysense and antisense DNA strands in a suitable buffer solution. In thissynthesis process, cohesive terminal sequences for cloning purpose canbe introduced to both ends of the oligonucleotide. Then theoligonucleotide encoding the peptide of the invention is linked by T4ligase to a suitable vector containing the sequence encoding anotherpolypeptide or protein digested by relevant restriction endonuclease toobtain the DNA sequence encoding the fusion protein. Generally, thecoding sequence for another polypeptide or protein is known. Some ofthem can be obtained from the market in the form of vectors, others canbe synthesized or cloned from a known organism based on conventionalmethods. The sequencing of the obtained gene sequence of the presentinvention or various DNA fragments can be performed by a conventionalmethod such as dideoxy chain-termination method (Sanger et al., PNAS,1977, 74: 5463-5467). The sequencing can also be performed by usingcommercial DNA sequencing kits. Of course, the expression vector shouldcontain an appropriate promoter, ribosome binding site, terminator andthe like. An appropriate leader sequence can be linked to the upstreamof the polypeptide-coding sequence in order to direct the expressedproduct to certain cell apartments. The selection of an appropriatevector and promoter is well known to an ordinary person skilled in theart. An effective vector for bacteria can be constructed as follows: thestructural DNA sequence encoding the target protein together with anappropriate initiation signal and termination signal for translation isinserted into an operable open reading frame containing a functionalpromoter. The methods for constructing a vector containing a nucleotidesequence of the present invention and appropriate regulatory elementsfor transcription and translation are well known to an ordinary personskilled in the art. It is known to those skilled in the art that, toexpress the target protein, an appropriate host should be selectedaccording to the type of the expression vector or construct into whichthe DNA sequence of the present invention has been inserted. Theappropriate hosts for the expression of the polypeptides of the presentinvention include, but not limited to, prokaryotic host such as E. coli,Bacillus, Streptomyces etc.; eucaryotic host such as Saccharomyces spp.,Aspergillus spp.; insect cell such as Drosophila S2 cell and Spodopterafrugiperda Sf9; mammalian cells such as CHO and COS (monkey renalfibroblast cell line, Gluzman (Cell 23:175, 1981)); other cell linescapable of expressing a compatible vector. The methods for introducing aconstruct into the above-mentioned host cells are well known to thoseskilled in the art, and include, but not limited to, calciumchloride-mediated transformation, calcium phosphate-mediatedtransfection, DEAE-dextran-mediated transfection, electroporation,micro-injection, particle bombardment method or gene gun method(Sambrook, J. (1989), Molecular Cloning, a Laboratory Manual, ColdSpring Harbor Press; Plainview, N.Y.; Ausubel, F. M. (1989) CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y.; Hobbs, S. etal., McGraw Hill Yearbook of Science and Technology (1992), McGraw Hill,N.Y. 191-196; Engelhard, E. K. et al., PNAS, 91:3224-3227; Logan, J. etal., PNAS, 81:3655-3659). The transformed host strains or cells arecultured in a suitable culture medium under suitable culture conditions.When they grow to appropriate cell density, the selected promoter may beinduced by an appropriate method (such as temperature transition orchemical induction). After the induction, the cells are cultured foranother period of time. The selection of culture conditions and mediacorresponding to different host strains or cells and the features of theexpressed target protein is within the knowledge of those skilled in theart. The expressed polypeptides or fusion proteins may be separated andpurified from the culture by a conventional method such ascentrifugation, precipitation, various chromatographies, HPLC, etc.Example 4 shows the cloning and expression process of a fusion proteinwhich is composed of a peptide of the present invention anddihydrofolate reductase.

The peptides of the present invention have anti-angiogenesis activity.As shown in the experimental examples, the peptides can competitivelyinhibit the binding of VEGF to its receptor Flt-1, specifically inhibitthe proliferation of angioendothelial cell, the proliferation of chickchorioallantoic membrane blood vessels, and the tumor growth andmetastasis in nude mice. As angiogenesis inhibitor, the peptides of thepresent invention are useful for the treatment of primary or metastaticsolid tumors and cancers of the following organs: breast, colon, rectum,lung, oropharynx, hypopharynx, esophagus, stomach, pancreas, liver,bilecyst, bile duct, small intestine; urinary system including kidney,bladder and epithelium of urinary tract; female genital system includingunerine neck, uterus, ovary, chorioma and gestational trophoblasticdiseases; male genital system including prostata, seminal vesicle andtestis; endocrine glands including thyroid gland, adrenal gland andpituitary body; skin cancer including angioma, melanoma, sarcomaoriginated from bone or soft tissue, and Kaposi's sarcoma; tumors ofbrain, nervus, eye and meninges, including astrocytoma,neuroastrocytoma, spongioblastoma, retinoblastoma, neuroma,neuroblastoma, neurinoma and neuroblastoma; solid tumors developed frommalignant diseases of hemopoietic system, including chloroleukemia,plasmacytoma and dermal T lymphoma/leukaemia; lymphoma includingHodgkin's lymphoma and non-Hodgkin's lymphoma. The peptides of thepresent invention can also be used to prevent autoimmune diseases,including rheumatoid arthritis, immune arthritis and degenerativearthritis; eye diseases, including diabetic retinopathy, Terry'ssyndrome, rejection of corneal implantation, retrolental fibroplasia,neovascular glaucoma, neovascularization of retina induced bydegeneration of macula and anoxia; dermatosis including psoriasis;angiopathy including hemangioma and capillary proliferation inarteriosclerosis plaques; angiogenesis in cardiac muscle;neovascularization of plaques; capillarectasia; hemorrhagic articularangiofibroma; granulation of wound; diseases characterized in excessiveor abnormal stimulation of endothelial cell, including regional ileitisof intestinal adhesion, arteriosclerosis, dermatasclerosis andhypertrophic scar; and other diseases associated with angiogenesisincluding ulcer. Additionally, the peptides of the present invention caninhibit ovulation and formation of placenta, therefore may be used asprocreation-controlling agents. Peptides of the present invention canalso be used in preventing metastasis of the above-mentioned tumors.

The compounds of the present invention can be used in the form ofpharmaceutically acceptable salts. A “pharmaceutically acceptable salt”refers to salt without undue toxicity, stimulation, allergy etc. whilecontacting human or animal tissues. Pharmaceutically acceptable saltsare well known in the art. The salts can be prepared during the finalseparation and purification process of the peptides of the presentinvention. The salts can also be prepared through the reaction of freebase or acid form with suitable organic or inorganic acid or base. Therepresentative acid addition salts include, but not limited to, acetate,adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate,bisulfate, butyrate, camphorate, camphorsulfonate, glycerophosphate,semisulfate, heptylate, hexoate, fumarate, hydrochlorate, hydrobromate,hydriodate, 2-hydroxyethyl-sulfonate, lactate, maleate,methane-sulfonate, nicotinate, 2-naphthalene-sulfonate, oxalate,3-phenyl-propionate, propionate, succinate, tartrate, phosphate,glutamate, bicarbonate, p-toluenesulfonate and undecylate. The preferredacids for forming pharmaceutically acceptable salts are hydrochloricacid, hydrobromic acid, sulfuric acid, phosphoric acid, oxatic acid,maleic acid, succinic acid and citric acid. The basic ions ofpharmaceutically acceptable base addition salts include, but not limitedto, alkali metal ions or alkaline earth metal ions such as lithium,sodium, potassium, calcium, magnesium and aluminum etc., and non-toxicquaternary ammonium such as ammonium, tetramethyl ammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine,diethylamine, ethylamine, diethylamide, ethanolamine, diethanolamine,piperidine and piperazine etc. The preferred base addition salts includephosphate, tris and acetate.

A pharmaceutical composition can be formulated by combining a peptide ora fusion protein of the present invention and a pharmaceuticallyacceptable excipient or carrier. Pharmaceutically acceptable carriers orexcipients refer to nontoxic fillers, diluents, encapsulation materialsor other adjuvants in the state of solid, semisolid or liquid. Thepharmaceutical compositions can be administered via parenteral,sublingual, intracisternal, intravaginal, intraperitoneal, intrarectal,intrabuccal, intra-tumor or topical routes.

The parenteral administration includes intravenous, intramuscular,intraperitoneal, intrasternal, subcutaneous, intra-articular injectionand transfusion. The pharmaceutical compositions suitable for parenteraladministration include sterilized aqueous solution or non-aqueoussolution, dispersion, suspension or emulsion, and powder to be dissolvedin injectable sterile solution or dispersion liquid immediately beforeuse. Suitable aqueous carriers or non-aqueous carriers, diluents,solvents or excipients include water, ethanol, glycerin, propyleneglycol, polyethylene glycol, carboxymethyl cellulose, vegetable oil andinjectable organic ester such as ethyl oleate. These compositions mayalso comprise adjuvants such as preservative, moisture agent, emulsifierand disperser etc. The addition of isotonic agent such as saccharide,sodium chloride may be advantageous.

The topical administration includes administrations on the surface ofskin, mucosa, lung and eye. The pharmaceutical compositions for topicaladministration include powder, ointment, drops, transdermal patch,iontophoresis apparatus and inhalant etc. In the inhalationalcompositions, the peptides of the present invention are compressed orcontained in a pressured gas propellant such as nitrogen or liquidizedgas propellant. The peptides of the present invention are preferably notsoluble in the liquidized propellant medium.

Preferably, the formulations for intrarectal or intravaginaladministration are suppository that can be formulated through mixing thepeptides of the present invention with appropriate non-stimulatingexcipients or carriers such as cacao butter, polyethylene glycol orsuppository wax. Said excipients or carriers are solid at roomtemperature, but liquid at body temperature, therefore can be melted inthe cavity of rectum or vagina releasing the active ingredients therein.

For administration by the above-mentioned routes or other routes, thetherapeutically effective amount of peptides of the present inventioncan be in the pure form or the form of pharmaceutically acceptable saltswith or without a pharmaceutically acceptable excipient. The term“therapeutically effective amount” refers to an amount of the peptidesof the present invention sufficient to exhibit a therapeutic effect onthe angiogenic diseases within a suitable therapeutic window. Thespecific therapeutically effective amount for a specific subject dependsupon many factors including the nature and severity of the disease to betreated; the activity of the specific compound used; the specificcomposition used; the subject's age, body weight, gender, diet andgeneral health status; administering time; administering route; theexcretion rate of the specific compound; duration of treatment; otherdrugs administered jointly or simultaneously. For example, the totaldaily dose of a peptide of the present invention may be 0.05-20 mg/kgbody weight when administered topically, 0.15-50 mg/kg body weight whenadministered systemically. The daily dose of a fusion protein having amolecular weight of 27,000 dalton may be 0.05-60 mg/kg body weight whenadministered topically, 0.5-150 mg/kg body weight when administeredsystemically. The daily dose can be divided and administered for severaltimes in one day if necessary. Therefore, the single dosage form of thecompositions can comprise the total daily dose or part of the same.

In another aspect, the present invention also relates to polynucleotidesencoding the polypeptides of the present invention as defined above.

The present invention also relates to gene therapy. That is, thetherapeutic effects are obtained through transferring the gene encodinga polypeptide of the present invention into a subject's cells to expressthe polypeptide in vivo. A variety of the methods for transferring ordelivering DNA to cells for expressing proteins are known in the art,for example in N. Yang, “In Vivo Gene Transfer in Mammal Somatocytes”,Crit. Rev. Biotechn. 12(4): 335-356(1992). Gene therapy includesincorporation of DNA sequences into somatocytes or germ cells for exvivo or in vivo therapy.

There are three main types of methods of gene transfer for gene therapy:(1) physical methods, such as electroporation, direct gene transfer andparticle bombardment, (2) chemical methods, such as methods lipid-basedvectors and other non-viral vectors, and (3) biological methods, such asviral vectors. For example, a non-viral vector such as liposomecontaining DNA can be intravenously injected to a subject.Alternatively, vectors or “naked” DNA gene can be injected into thetarget organ, tissue or tumor directly to deliver the therapeutic DNA tothe target.

The basic methods of gene transfer include ex vivo gene transfer, invivo gene transfer and in vitro gene transfer. For ex vivo genetransfer, the cells are obtained from a specific subject and cultured ina cell medium. Then a DNA is transfected into the cells. The transfectedcells are expanded and are re-transplanted into the same subject. For invitro gene transfer, the cells to be transformed are the cells culturedin a medium such as cultured tissue cells instead of specific cells froma specific subject. These “Laboratory Cells” are transfected. Thetransfected cells are selected and cultured to proliferate, and thentransplanted into a subject or used for other purposes. In vivo genetransfer involves DNA transfer into the cells which maintain in the bodyof a subject.

The biological gene therapy is preferred in the present invention, thatis, the gene is inserted into cells by means of a viral vector. As usedherein, the term “vector” refers to a vector which contains or bindswith a certain polynucleotide sequences, and is responsible fordelivering these polynucleotide sequences into the cells. The cells tobe transfected can be obtained from a normal tissue or a focus tissue ofa subject or not from a specific subject. The examples of vectorsinclude plasmid, infectious microbe like virus, or non-viral vectorssuch as ligand-DNA conjugate, liposome and lipid-DNA complex.

A recombined DNA molecule containing the DNA sequence encoding thepeptide or fusion protein of the present invention is effectively linkedto a regulatory sequence to form an expression vector which can expressthe polypeptides or the fusion proteins of the present invention. Theviral vectors which have been used in gene therapy include, but notlimited to, retrovirus, other RNA virus such as poliomyelitis virus orsindbis virus, adenovirus, adeno-associated virus, herpes virus, simianvirus 40, poxvirus or other DNA virus. Murine replication defectiveretrovirus vector is the most popular vector for gene transfer. Viralvectors have also been used to insert gene into cells in vivo. Toexpress a heterogenous gene in target tissue specifically, a knowntissue-specific cis-regulatory element or promoter can be used.Alternatively, DNA in situ delivery or anatomical site-specific viralvectors can also be used to achieve the same effect. For example, thegene transfer to vessels has been achieved by transplantation ofendothelial cells from a chosen site of the artery wall which aretransfected in vitro. Surrounding cells infected by the virus can alsoexpress the gene products. Alternatively, viral vectors can be deliveredto a site in vivo directly (for example through catheter), thereby onlya few sites are infected by the virus and long-term site-specific geneexpression is provided. In vivo gene transfer in mammary tissue andhepatic tissue has been achieved using retrovirus vectors wherein therecombined virus is injected to the vessels directing to these organs.

Accordingly, the present invention further relates to a plasmid or viralvector containing a polynucleotide of the present invention, and a cellcontaining a polynucleotide or vector of the present invention.

As mentioned above, the present invention relates to a polynucleotideencoding a polypeptide of the present invention, vector or cellcontaining the polynucleotide used as gene therapeutical medicament.

Correspondingly, the present invention relates to a pharmaceuticalcomposition for treating angiogenic diseases. The composition comprisesa polynucleotide, vector or cell of the present invention, and apharmaceutically acceptable excipient, especially one suitable for genetherapy if required.

Based on the use of a polynucleotides of the present invention in genetherapy, the present invention relates to use of a polynucleotide orvector as defined above for the preparation, from cells obtained from asubject, of cells to be re-transplanted to the subject for treatingangiogenic diseases.

Finally, the present invention relates to a method for treatingangiogenic diseases comprising administering to a subject in need suchtreatment a therapeutically effective amount of a peptide,polynucleotide, vector or cell as defined in the present invention.

The present invention is further illustrated by the following exampleswhich however are not intended by any way to restrict the protectionscope of the present invention.

EXAMPLE 1

H₂N-Trp-His-Ser-Asp-Met-Glu-Trp-Trp-Tyr-Leu-Leu-Gly-COOH (F56) (SEQ IDNO: 1).

Peptide synthesis column packed with4-(2′,4′-dimethoxyphenyl-Fomc-aminomethyl)phenoxylacetamidoethyl resinwas loaded in a Pioneer™ peptide synthesizer. The peptide wassynthesized in nitrogen according to the following steps:

1. The resin was solvated in DMF for 5 minutes;

2. The resin was treated with 20% piperidine in DMF for 15 minutes toremove the protective group Fmoc on the grafted group of the resin (oron α-amino group of the amino acid attached to the resin);

3. The resin was rinsed by DMF for several minutes;

4. The α-carboxy group of the first amino acid Gly (Fmoc-Gly) of Cterminal was activated with 0.2 M solution of HBTU and HOBT in DMSO-NMP(N-methyl-pyrrolidone) and 0.4 M diisopropylethylamine solution;

5. The activated amino acid obtained in step 4 was coupled with theresin of step 2 (or the amino acid attached to the resin) in DMF forabout 30 minutes;

6. The resin was rinsed by DMF for 5 minutes;

7. Steps 2-6 were repeated with the following amino acids:

-   Fmoc-Leu→Fmoc-Leu→Fmoc-Tyr(tBu)→Fmoc-Trp→Fmoc-Trp→Fmoc-Glu(γ-OtBu)→Fmoc-Met→Fmoc-Asp(β-OtBu)→Fmoc-Ser(tBu)→Fmoc-His(Trt)→Fmoc-Trp;

8. The resin was rinsed with THF for about 5 minutes;

9. The resin and a fresh cleavage mixture (thianisole:waterehtanedithiol:trifluoroacetic acid=2:1:1:36, by volume) were mixed andstirred for 10-15 minutes at 0° C., and then stirred for 2 hours at roomtemperature;

10. The reaction mixture was filtrated and the filtrate was centrifugedin cold ethyl ether. The supernatant was poured out and centrifuged incold ethyl ether again until the peptide was fully precipitated. The rawpeptide was purified by chromatography on a preparative C18 silica gelcolumn. The column was eluted gradiently by acetonitrile/(water, 0.1%TFA). The fractions containing the target peptide were collected andlyophilized to obtain 30 mg of the title peptide.

EXAMPLE 2

H₂N-Trp-His-Val-Asp-Glu-Thr-Trp-Trp-Leu-Leu-Met-Leu-COOH (F87) (SEQ IDNo: 2).

The title peptide was prepared according to the procedure described inExample 1. The first amino acid was Fomc-Leu. The following amino acidswere added according to the following order under said conditions:Fmoc-Met→Fmoc-Leu→Fmoc-Leu→Fmoc-Trp→Fmoc-Trp→Fmoc-Thr(tBu)→Fmoc-Glu(γ-OtBu)→Fmoc-Asp(β-OtBu)→Fmoc-Val→Fmoc-His(Trt)→Fmoc-Trp.35 mg of the title peptide was obtained.

EXAMPLE 3

H₂N-Trp-His-Asp-Pro-Thr-Pro-Trp-Trp-Ser-Trp-Glu-Ile-COOH (F90) (SEQ IDNO: 2).

The title peptide was prepared according to the procedure described inExample 1. The first amino acid was Fomc-Lle. The following amino acidswere added according to the following order under said conditions:Fmoc-Glu(γ-OtBu)→Fmoc-Trp→Fmoc-Ser(tBu)→Fmoc-Trp→Fmoc-Trp→Fmoc-Pro→Fmoc-Thr(tBu)→Fmoc-Pro→Fmoc-Asp(β-OtBu)→Fmoc-His(Trt)→Fmoc-Trp.44 mg of the title peptide was obtained.

EXAMPLE 4 Preparation of Fusion Protein DHFR-F56/DHFR-F90

1. Preparation of the DNA Encoding Peptides

According to a DNA sequence encoding peptide F56 or F90, sense andantisense single strand DNA fragments were synthesized on a DNAsynthesizer (ABI, 318A type) by the solid phase phosphite triestermethod. Cohesive terminal sequences for cloning were added to both endsof the single strand DNA fragments. 30 ng of each of sense and antisenseDNA were incubated in 8 μl TE in water bath at 65° C. for 5 minutes, andcooled down to room temperature in 15-30 minutes gradually.

2. Construction of DHFR-F56/DHFR-F90 Expression Vector and InducedExpression on Small Scale

The expression plasmid for dihydrofolate reductase, pQE 42, was digestedby Pst I and Hind III (purchased from New England Biolabs) in turn andretrieved by Qick kit (product of QIAGEN company). The purified vectorfragment was linked to the annealed oligonucleotide fragment obtainedaccording to the above 1) by T₄ DNA ligase (purchased from New EnglandBiolabs). Due to the anti-ampicillin activity of E. coli M15, 1 μllinkage product was transferred into JM109 by electroporation first, andthe positive clones were selected by LB (1.5% agar, 100 μg/mlampicillin) medium. Then the plasmids were amplified and identified byBamH I digestion. The method of the identification and the principlethereof are as follows: each of the DNA sequences of F56, F90 has adigestion site of BamH I. There is also a digestion site of BamH I onthe vector DNA fragment close to the Pst I site. Therefore, a DNA bandof about 700 bp can be obtained through digestion of the constructed pQE42-F56/F90 by BamH I , but not from the same digestion of the emptyvector without cloning fragment inserted in. The results of theidentification by digestion were totally coincident with the principle,indicating that F56 and F90 have been cloned into the expression vectorpQE42.

The right plasmid pQE42-F56/F90 identified by digestion was transformedinto E. coli M15. The transformed products were screened by being spreadon LB culture dishes (1.5% agar, 100 μg/ml ampicillin, 25 μg/mlkanamycin). The positive clones were selected and cultured in LB (100μg/ml ampicillin, 25 μg/ml kanamycin), and protein expression wasinduced by IPTG (0.1 mmol/L). The expressed products were analyzed by10% SDS-PAGE.

3. Large Scale Expression and Purification of DHFR-F56/DHFR-F90 FusionProtein

The bacteria pellet from 100 ml culture broth of large scale inducedexpression was suspended in 2 ml TE solution and incubated in ice bath.The bacteria were lysed by intermittent ultrasound for 30 s andcentrifuged (12,000g, 10 minutes, 4° C.). The supernatant was discarded.The pellet was suspended in 2 ml sodium deoxycholate (20 g/L) byultrasound and incubated at room temperature for 30 minutes. Thesuspension was centrifuged (10,000 g, 10 minutes, 4° C.) to obtain theinclusion body as pellet. A certain volume of denaturing solution wasadded to the inclusion body and incubated at room temperature for 3hours. Then, 10× volume of renaturing solution was added slowly. Afterthe solution was incubated at room temperature for 3-4 hours, its pHvalue was adjusted to 8. The solution then was centrifuged (10,000 g, 4°C., 10 minutes). The purity and content of the protein was analyzed by10% SDS-PAGE.

4. Western Blot Assay

Protein DHFR (positive control), DHFR-F56, DHFR-F90 and 6His-Kringle(I-V) (negative control) were loaded onto 10% SDS-PAGE to performelectrophoresis. Murine monoclonal antibody specific for DHFR preparedby the inventor's laboratory was used as the primary antibody.Horseradish peroxidase labeled rabbit-anti-mouse antibody was used asthe secondary antibody. The binding of DHFR specific monoclonal antibodyto DHFR, DHFR-F56, DHFR-F90, 6-His-Kringle (I-V) were analyzed byWestern blot. The analysis results of the fusion proteins obtained inthe present example were positive.

EXMAPLE 5 Binding of DHFR-F56/DHFR-F90 to sFlt-1 and Endothelial Cell

The specificity of the binding of the fusion proteins DHFR-F56 andDHFR-F90 to receptor Flt-1 was tested by ELISA in the present example.

Particularly, a 96-well plate was coated with soluble receptors, sFlt-1,sKDR (5 mg/L, 50 μl/well) [Song Shumei, Shou Chengchao: Molecularcloning and its expression of vascular endothelia growth factor receptorKDR. Chin J. Biochem. Mol. Bio. 1998, 14 (1): 57-61; An Ping, DongZhewei and Shou Chengchao: Molecular cloning and its bio-activity ofsoluble VEGF receptor expressed in bacterial. Chin J. Biochem. Mol. Bio.2000, 16 (1): 62-66]. Alternatively, the vascular endothelial cells in a96-well plate were fixed by pre-cooled 0.125% glutaraldehyde solution.DHFR, DHFR-F56 or DHFR-F90 was added to the plate after blocking. AnELISA assay was carried out using a DHFR-specific monoclonal antibodyaccording to conventional method. The ELISA assay was repeated for 3times.

The results are shown in the following table 1. The results show thatDHFR-F56 and DHFR-F90 can bind to Flt-1and endothelial cells, and haveno notable reaction with KDR. The negative control, DHFR, has noreaction with sFlt-1, KDR or endothelial cells. These results indicatethat the binding of DHFR-F56/DHFR-F90 to sFlt-1 and endothelial cells isspecific.

TABLE 1 Test for the specificity of the binding of DHFR-F56/DHFR-F90 byELISA (OD₄₉₂ nm) DHFR-F56 DHFR-F90 DHFR SFlt-1 0.529 0.415 0.088 SKDR0.081 0.074 0.074 EC 0.477 0.384 0.080

EXAMPLE 6 Inhibition of the Binding of VEGF to Receptor Flt-1 byDHFR-F56

In order to detect the activity of DHFR-F56 blocking VEGF binding tosFlt-1, a binding assay was carried out using ¹²⁵I-VEGF (purchased fromLife Technology Inc.).

The experiment was carried out according to a method substantially asdescribed by Tanaka, K., et al (Characterization of the extracellulardomain in vascular endothelial growth factor receptor-1 (Flt-1 tyrosinekinase). Jpn J Cancer Res. 1997, 88:867-876). Briefly, a 96-well platewas coated overnight at 4° C. by 0.5 mg/L soluble receptor of VEGF,Flt-1 (sFlt-1). The coating solution was 0.01 mol/L phosphate buffer (pH6.2). Then, the plate was rinsed with a washing solution (0.9% NaCl,0.01 mol/L, pH6.2) for 3 times. 250 μl blocking buffer (0.3% BSA, 25mmol/L HEPES, DMEM, pH7.6) was added into each well for 3 hours at roomtemperature. DHFR-F56 of different concentrations (1, 5, 10, 15, 20μg/ml) (DHFR of different concentrations for the control group) and¹²⁵I-VEGF (purchased from Life Technology Inc.) of fixed concentration(2 μg/L, 50 μg/well) were added to the wells. 2 parallel wells were usedfor each concentration. The binding reaction underwent for 1 hour atroom temperature. The plate was rinsed 4 times with the binding buffer.After the 96-well plate was dried completely, a liquid-scintillationsolution was added into the plate (120 μl/well). The cpm value wasdetermined by a Wallac co liquid-scintillation counter after the solidresidue had been dissolved.

The results are shown in FIG. 1. The binding of ¹²⁵I-VEGF to coatedsFlt-1 decreases along with the increase of the concentration ofDHFR-F56, while the, change of the concentration of DHFR has no notableeffect on the binding of ¹²⁵I-VEGF to sFlt-1. There is statisticallysignificant difference between DHFR-F56 group and DHFR group (hypothesistesting of paired data comparison, t test, α=0.05). This indicates thatF56 can competitively inhibit the binding of VEGF to receptor Flt-1.

EXAMPLE 7 Effect of Peptide F56/F90 to the Proliferation of EndothelialCells

A sample of human umbilical cord (obtained from Beijing Gynecologicaland Obstetric Hospital) was digested by 0.1% collagenase II (purchasedfrom GIBCO BRL Corporation), and centrifuged at 1000 rpm for 10 minutesto collect the human umbilical cord vein-derived endothelial cells(HUVECs). The cells were suspended in 20% FCS/RPMI 1640 (purchased fromGIBCO BRL Corporation), and transferred to a plastic culture flask andcultured in a incubator (37° C., 5% CO₂) for 2 days. Then, the culturedHUVECs were digested with 0.04% trypsin-EDTA, and inoculated into a24-well plate at 3×10⁴ cells/well. After 24 hours' incubation, the cellswere incubated for another 24 hours in new medium 2% FCS/RPIM 1640.VEGF₁₆₅ (product of Sigma Corporation) was added either independently oras a mixture with peptide F56 or F90 of different concentrations (50,100, 200, 300 μmol/L). 4 parallel wells were used for each group. Thecells were incubated for 48 hours. Then, ³H-TdR(1 μci/ml) was added andthe cells were incubated for another 8 hours. The cells were collectedto a glass-fiber filter membrane. The cpm value was detected accordingto conventional methods. The same experiment was repeated for 3 times.

The results are shown in FIG. 2. Peptide F56/F90 has no significantinhibition to the proliferation of umbilical cord vein-derivedendothelial cells. This is consistent with the theory that the bindingof VEGF to Flt-1 does not result in the proliferation of endothelialcells.

EXAMPLE 8 Inhibition of the Proliferation of Blood Vessels on ChickChorioallantoic Membrane by F56

In this example, a proliferation assay on chick chorioallantoic membraneblood vessels was carried out to detect the inhibition of F56 toangiogenesis.

The experiment was carried out according to a method described by FuShengfa et al. (Fu Shengfa, Lu Yinglin and Zhang Chaoshan: Technique ofdetecting the effects of vascular growth factor with chorioallantoicmembrane of fertilized chicken eggs. Chin Mili. Acad. Medi. Scie. 1993,17:294-97”). Particularly, fresh inseminated chicken eggs were soaked in0.1% benzalkonium bromide solution for 2 minutes at 38° C., andincubated in an incubator at 37.5° C., 60-70% humidity for 3 days. Then,the eggs were stripped and cultured on a petri dish. When the allantoicmembrane grew to about 1cm², the different groups (5 chick embryos pergroup) were treated with the following agents. VEGF₁₆₅ (2 μg/L), DHFR(negative control) or 18 μl F56 solution (0.8 μg/μl) was dropped onto aglass-fiber filter membrane (Φ5 mm). The membrane was placed on thedistal end (in relation to the heart of the embryo) of thechorioallantoic membrane. The agents were administered once in themorning and once in the evening for 2 days. The results were observedand recorded by photography. The experiment was repeated twice (Kodak100, exposed for 3 s).

The results are shown in FIG. 3. The results indicate that humanVEGFI₁₆₅ can promote the proliferation of chick chorioallantoic membraneblood vessels (FIG. 3A). While F56 can lead to the rupture of bloodvessels and bleeding in addition to blood vessel atrophy (FIG. 3C). DHFRhas no notable effect on the proliferation of chick chorioallantoicmembrane blood vessels (FIG. 3B). This indicates that F56 has thebiological activity of anti-angiogenesis.

EXAMPLE 9 Inhibition of Tumor Growth in Nude Nice by DHFR-F56/DHFR-F90

To testify whether DHFR-F56 and DHFR-F90 can inhibit tumor growth on thebasis of anti-angiogenesis, a tumor inhibition test on Balb/c nude micewas performed.

MGC803 cells (a human gastric carcinoma cell line) was subcutaneouslyimplanted on the right buttock of eight 5-week aged nude mice (purchasedfrom the Animal Center of the Chinese Academy of Medical Sciences)(2×10⁶ cells/0.1 ml/each mouse). On the fifth day after theimplantation, the nude mice were grouped at random into 2 groups each ofwhich included 4 mice. DHFR (150 μg/100 μl per time), DHFR-F56 orDHFR-F90 (150 μg/100 μl per time) was injected intraperitoneally everyother day for 10 times. The nude mice were observed for 30 days afterwithdrawal. The major and minor axes of the tumors were measured byvernier caliper every three days during the observation period. Thevolume of the tumor was calculated according to the formula of ½×majoraxis×minor axis². After the 30 days, the nude mice were put to dead byneck-breaking. The tumors were weighted and analyzed statistically.

The results are shown in FIG. 4 and FIG. 5. Both DHFR-F56 and DHFR-F90can inhibit the growth of human gastric carcinoma MGC803 cells in Balb/cnude mice, and slow the growth rate of tumor. The statistical analysis(two-sample t test) indicates that the tumor volume of treatment groupis smaller generally. There is significant difference between thetreatment group and the DHFR control group (p<0.05). Furthermore,DHFR-F56 can also result in necrosis of tumor (FIG. 4).

EXAMPLE 10 Inhibition of Cancer Metastasis by F56

In order to observe the inhibitory activity of the peptides of thepresent invention to cancer metastasis, a metastasis model of breastcancer in SCID mice as described by Li Zhihong at al. (Transplant ofhuman breast cancer in nude mice. Natl. Med J Chin. Vol.80 (11), 2000)was used to determine the function of F56. Briefly, human breast cancercells were inoculated under the fat pad in the breast of SCID mice.Peptide F56 (0.5 mmol/L) was injected topically (60 μl per mouse pertime) on the next day and every other day for 10 times in total. After 8weeks, the mice were put to dead. The tumors were taken out andweighted. The whole lung was taken out, fixed and sliced with maximalcross-section and dyed by HE. The number of metastatic foci was counted.The results as shown in table 2 indicate that F56 can significantlyinhibit cancer metastasis to lung in addition to the inhibition of tumorgrowth on the injection sites.

TABLE 2 F56 inhibited the metastasis of breast cancer cells to lungs ofSCID mice Number of metastatic foci in each mouse Total MetastasisPeptide 1 2 3 4 5 number Mean inhibition (%) F56 0 0 1 2 2 5 1.0 70.5Solvent 5 4 3 2 3 17 3.6 0

1. A synthetic polypeptide having the amino acid sequence of SEQ IDNO.:1, and pharmaceutically acceptable salts thereof.
 2. A fusionpolypeptide comprising the polypeptide of claim 1 and dihydrofolatereductase.
 3. A pharmaceutical composition comprising a polypeptide ofclaim 1 and a pharmaceutically acceptable carrier or excipient.
 4. Amethod of treating disease marked by increased angiogenesis comprisingadministering to a subject with said disease a therapeutically effectiveamount of a polypeptide of claim 1.